Apatite surface neutralization with alkali solutions

ABSTRACT

The present invention discloses methods of neutralizing apatite surfaces, for example after a flow-through collection of a target and prior to cleaning the chromatography solid support.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims benefit of priority to U.S. ProvisionalPatent Application No. 61/438,729, filed Feb. 2, 2011, which isincorporated by reference.

BACKGROUND OF THE INVENTION

Hydroxyapatite and fluorapatite, among other apatite solid supports, areused for purification of a wide variety of biomolecules, includingproteins, carbohydrates, polynucleotides, and viral particles.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for a method for cleaning an apatitesolid surface following target molecule purification by a non-adsorbingflow through process. In some embodiments, the method comprises,

(a) contacting a sample comprising the target molecule to an apatitesolid surface thereby flowing the target molecule past the apatite solidsurface;(b) neutralizing the apatite solid surface by contacting the apatitesolid surface with a sufficient concentration and volume of an alkalinehydroxide; and(c) cleaning the apatite solid surface.

In some embodiments, the alkaline hydroxide is selected from the groupconsisting of sodium hydroxide, potassium hydroxide and lithiumhydroxide.

In some embodiments, the concentration of the alkaline hydroxide isbetween 0.1 and 1 M. In some embodiments, the concentration of thealkaline hydroxide is between 0.3 and 0.7 M.

In some embodiments, the cleaning comprises contacting the solid surfacewith a phosphate solution. In some embodiments, the phosphate solutionhas a pH at or between 6.5 and 10.0. In some embodiments, the phosphateconcentration of the phosphate solution is at or between 0.1 and 1.0.

In some embodiments, the apatite is selected from the group consistingof hydroxyapatite and fluorapatite. In some embodiments, the apatite isceramic hydroxyapatite or ceramic fluorapatite.

In some embodiments, the apatite is a non-ceramic apatite.

In some embodiments, the target molecule is a protein. In someembodiments, the protein is an antibody.

In some embodiments, the contacting comprises contacting the solidsurface with a solution at a pH of between 5.0 and 7.5.

In some embodiments, the apatite solid support is in the form of acolumn.

Definitions

“Neutralizing the solid apatite surface” refers to treating the surfaceof the apatite surface such that the solid surface does not containsufficient hydronium ions to significantly affect (i.e., cause a greaterthan 0.2 acidic pH shift of) the pH of a subsequent cleaning buffer.

“Antibody” refers to an immunoglobulin, composite, or fragmentary formthereof. The term may include but is not limited to polyclonal ormonoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM,derived from human or other mammalian cell lines, including natural orgenetically modified forms such as humanized, human, single-chain,chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitrogenerated antibodies. “Antibody” may also include composite formsincluding but not limited to fusion proteins containing animmunoglobulin moiety. “Antibody” may also include antibody fragmentssuch as Fab, F(ab′)2, Fv, scFv, Fd, dAb, Fc and other compositions,whether or not they retain antigen-binding function.

An “apatite solid surface” refers to fused nanocrystals (ceramicapatite), microcrystals, or compounded microcrystals. Ceramic apatitesinclude, but not limited to, ceramic hydroxyapatite (e.g., CHT™) orceramic fluorapatite. Ceramic apatites are a form of apatite minerals inwhich nanocrystals are agglomerated into particles and fused at hightemperature to create stable ceramic microspheres suitable forchromatography applications. Compounded microcrystals include but arenot limited to HA Ultragel® (Pall Corp.). Microcrystals include but arenot limited to Bio-Gel HTP, Bio-Gel® HT, DNA-Grade HT (Bio-Rad) andHypatite C (Clarkson Chromatography).

“Hydroxyapatite” refers to a mixed mode solid support comprising aninsoluble hydroxylated mineral of calcium phosphate with the structuralformula Ca₁₀ (PO₄)₆(OH)₂. Its dominant modes of interaction arephosphoryl cation exchange and calcium metal affinity. Hydroxapatite iscommercially available in various forms, including but not limited toceramic, crystalline and composite forms. Composite forms containhydroxyapatite microcrystals entrapped within the pores of agarose orother beads.

“Fluorapatite” refers to a mixed mode support comprising an insolublefluoridated mineral of calcium phosphate with the structural formulaCa₁₀ (PO₄)₆F₂. Its dominant modes of interaction are phosphoryl cationexchange and calcium metal affinity. Fluorapatite is commerciallyavailable in various forms, including but not limited to ceramic andcrystalline composite forms.

“Sample” refers to any composition having a target molecule or particleof interest. A sample can be unpurified or partially purified. Samplescan include samples of biological origin, including but not limited toblood, or blood parts (including but not limited to serum), urine,saliva, feces, as well as tissues.

An “alkaline hydroxide” refers to a metal alkali hydroxide comprisingany cation elements in Group I of the periodic table, including, e.g.,lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs),and francium (Fr). Thus, exemplary alkaline hydroxides include, forexample, NaOH, LiOH, and KOH.

“Flow-through mode” refers to an operational approach to chromatographyin which the chromatography conditions are established so that a targetmolecule (from a sample) to be purified flows past the chromatographysupport upon application, while at least some other components of thesample are selectively retained, thus achieving removal of at least somenon-target components of the sample.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, on the surprising discoverythat an alkaline hydroxide solution (e.g., NaOH) is useful to neutralizethe surface of an apatite solid support following a flow throughpurification of a target molecule and before a cleaning step. Hydrogen(or hydronium) ions can accumulate on an apatite solid surface followingflow through purification of a target molecule. Surprisingly, if oneperforms a subsequent cleaning step (e.g., with a 0.1-1.0 M phosphatesolution) without first neutralizing the column, degradation of thecolumn can occur by displacement of calcium ions in the apatite support.By neutralizing the solid support with an alkaline hydroxide solutionprior to the cleaning step, one can avoid significant degradation thatcan otherwise occur to the apatite solid surface.

Initially, the sample containing the target molecule is contacted to theapatite surface in flow-through mode as is known in the chromatographyarts. In some embodiments, the flow through comprises a solution of pHat or between 5.0 and 7.5. Exemplary buffers include, e.g., phosphatebuffers, optionally also containing sodium (e.g., NaCl). Flow throughmay be conducted at fast linear flow rates such as 300-600 cm/hr.However slower linear flow rates such as 50-200 cm/hr are alsoapplicable.

Optionally, the apatite surface is previously sanitized and/orequilibrated prior to adsorption of the target to the surface. After thetarget molecule is run past the solid support (e.g., though a column),the solid support is neutralized prior to cleaning.

“Flow-through mode” as it relates to the invention herein, refers to anoperational approach to chromatography in which the buffer conditionsare established so that intact non-aggregated target to be purifiedflows through the chromatography support, while other molecules (e.g.,in some embodiments aggregates and other large molecules (includingviruses) are selectively retained, thus achieving their removal.Flow-through mode conditions can be developed depending on the specifictarget desired. Without intending to limit the scope of the invention,the following description is provided as a guide for developingflow-through conditions as desired for a particular protein. Anexemplary flow-through condition is, for example: Condition the columnfor flow though by sanitizing with 0.5-1.0 N NaOH, wash with 0.2 Msodium phosphate at pH 6.5-7.5, equilibrate the column with flow throughbuffer (5-20 mM sodium phosphate, pH 5.0-7.5), apply the sample andcollect the flow though containing the target molecule, wash the columnwith 0.3-1 column volume of 1 M NaOH, and clean the column with 0.1-1Mphosphate. However, any flow-through conditions are contemplated for theinvention.

Neutralization occurs after the target has flowed through and optionallybeen collected. The neutralization comprises contacting the apatitesurface with a sufficient amount of a solution comprising a sufficientconcentration of an alkaline hydroxide. Exemplary alkaline hydroxidesinclude, for example, NaOH, LiOH, and KOH, though other alkalinehydroxides can also be used as desired. In some embodiments, thealkaline hydroxide that neutralizes the apatite solid surface isbetween, e.g. , 1-100 mM, 1-20 mM, 10-2000 mM, 10-1000 mM, 10-500 mM,10-200 mM, or 10-100 mM, etc.

Instead of alkaline hydroxides, it is also believed that aminofunctional bases and alkaline carbonates can be used to neutralize theapatite surface as described herein for alkaline hydroxides. For exampleit is believed that neutralization can be achieved with any aminofunctional base (triethyamine, Tris, ammonia, etc.). However the aminocompound may form gas with the cleaning buffer and cause an ammonia-likeodor. It is also believed that alkaline carbonates (e.g., lithium,sodium or potassium carbonate) will neutralize the apatite surface.However, CO₂ gas could form in the column causing back pressure.

Neutralization of the apatite surface can be readily measured. Forexample, one can monitor the pH of the chromatography effluent followingthe alkaline hydroxide treatment. A neutral apatite surface will resultin a pH change of no more than 0.1 or 0.2 between the input and effluentfollowing neutralization. For example, if the pH of the cleaning bufferis input at 7.0, the effluent would not drop to less than 6.8 duringcleaning if the surface were neutralized. Alternatively, one can monitorcalcium ions in the effluent to determine whether the surface isneutralized. In the presence of released free hydronium ion, apatitereleases calcium. Thus, the presence of more calcium in the effluentthan was in the input buffer indicates that the surface has not beenneutralized.

An exemplary cleaning solution is a phosphate buffer of about 0.1-1.0 Mand having a pH of about 6.5-10.0. However, any cleaning conditions arecontemplated for the invention. The buffer may optionally also includeother salts (e.g., KCl, NaCl), though salts are not generally necessaryonce the surface has been neutralized.

II. Apatites

Those of skill will appreciate that a number of types of apatite solidsurfaces can be used in the invention. Commercial examples of ceramichydroxyapatite include, but are not limited to CHT Type I and CHT TypeII. Commercial examples of ceramic fluorapatite include, but are notlimited to CFT™ Type I and CFT Type II. Unless specified, ceramichydroxyapatite and ceramic fluorapatite refer to roughly sphericalporous particles of any average diameter, including but not limited toabout 10, 20, 40, and 80 microns. The choice of hydroxyapatite orfluorapatite, the type, and average particle diameter can be determinedby the skilled artisan. Other non-ceramic types of apatite solidsurfaces (including those sold as “gels”) can also be used according ofthe invention. Examples of non-ceramic solid apatites include but arenot limited to compounded microcrystals (e.g., HA Ultragel® (PallCorp.)) and microcrystals (e.g., Bio-Gel HTP, Bio-Gel® HT, DNA-Grade HT(Bio-Rad) and Hypatite C (Clarkson Chromatography)).

In preparation for contacting the sample with the apatite support, thechemical environment inside the column is typically equilibrated. Thiscan be accomplished, for example, by flowing an equilibration bufferthrough the column to establish the appropriate pH; conductivity;identity, molecular weight, and other pertinent variables.

In some embodiments, the sample preparation is also equilibrated toconditions compatible with the column equilibration buffer. In someembodiments, this involves adjusting the pH of the sample preparationprior to loading.

In some embodiments, after the column and sample preparation isequilibrated, the sample preparation is contacted with the column. Thesample preparation can be applied at a linear flow velocity in the rangeof, for example, about 50-600 cm/hr. Appropriate flow velocity can bedetermined by the skilled artisan.

In some embodiments, the invention is practiced in a packed bed column,a fluidized/expanded bed column and/or a batch operation where thesupport is mixed with the sample preparation for a certain time. In someembodiments, an apatite support is packed in a column. In someembodiments, the apatite support is packed in a column of at least 5 mminternal diameter and a height of at least 25 mm.

Another embodiment employs the apatite support, packed in a column ofany dimension to support preparative applications. Column diameter mayrange from less than 1 cm to more than 1 meter, and column height mayrange from less than 1 cm to more than 30 cm depending on therequirements of a particular application. Appropriate column dimensionscan be determined by the skilled artisan.

After use, the mixed mode column can optionally be cleaned, sanitized,and stored in an appropriate agent, and optionally, re-used. Indeed, onebenefit of the neutralization solution of the present invention is thatdegradation of an apatite column can be avoided or delayed. Thus, insome embodiments, one can use the column for ten or more times, e.g.,more than 20, more than 30, more than 40 or more than 50 cycles ofpurification.

III. Uses

The methods of the invention can be used to purify essentially anytarget molecule in a complex sample. In some embodiments, the targetmolecule to be purified is a component of a biological sample. Examplesof such components include but are not limited to proteins, lipids,sugars, carbohydrates, viral particles, amino acids, nucleic acids, andcan include combinations thereof, e.g., a lipidated or glycosylatedprotein, or mixtures thereof. In some embodiments, samples to which themethod is applied include unpurified or partially purified biomoleculesfrom natural, synthetic, or recombinant sources. Unpurified samples canbe derived from, e.g., plasma, serum, ascites fluid, milk, plantextracts, bacterial lysates, yeast lysates, or conditioned cell culturemedia. In some embodiments, partially purified samples come fromunpurified preparations that have been processed by at least onechromatography, ultrafiltration, precipitation, other fractionationstep, or any combination thereof. An exemplary target molecule is anantibody (including but not limited to a monoclonal antibody and/orantibody fragments) or other peptide or polypeptide. The chromatographystep or steps can employ any method, including but not limited to sizeexclusion, affinity, anion exchange, cation exchange, protein Aaffinity, hydrophobic interaction, immobilized metal affinitychromatography, or mixed-mode chromatography. The precipitation step orsteps can include, for example, salt or PEG precipitation, orprecipitation with organic acids, organic bases, or other agents. Otherfractionation steps can include but are not limited to crystallization,liquid:liquid partitioning, or membrane filtration. Ultrafiltration caninclude direct concentration of the sample and/or diafiltration.

EXAMPLE

The following example are offered to illustrate, but not to limit theclaimed invention.

A chromatography column of apatite is equilibrated with 5 mM sodiumphosphate buffer, pH 6.5. A sample solution of equilibration buffer orsample buffer containing a target molecule is applied to the apatitecolumn and purification of the target molecule achieved by flowing itthrough the apatite. Adsorbed hydrogen ion is neutralized by eluting thecolumn with sufficient amount of a strong base such as sodium, potassiumor lithium hydroxide. Finally, the apatite is cleaned with a phosphatebuffer of sufficient concentration to elute adsorbed biologicalcompounds such as DNA, basic proteins and endotoxin, etc.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method for cleaning an apatite solid surface following targetmolecule purification by a non-adsorbing flow through process, themethod comprising, (a) contacting a sample comprising the targetmolecule to an apatite solid surface thereby flowing the target moleculepast the apatite solid surface; (b) neutralizing the apatite solidsurface by contacting the apatite solid surface with a sufficientconcentration and volume of an alkaline hydroxide; and (c) cleaning theapatite solid surface.
 2. The method of claim 1, wherein the alkalinehydroxide is selected from the group consisting of sodium hydroxide,potassium hydroxide and lithium hydroxide.
 3. The method of claim 1,wherein the concentration of the alkaline hydroxide is between 0.01 and2 M.
 4. The method of claim 1, wherein the concentration of the alkalinehydroxide is between 0.1 and 1 M.
 5. The method of claim 1, wherein thecleaning comprises contacting the apatite solid surface with a phosphatesolution.
 6. The method of claim 5, wherein the phosphate solution has apH at or between 6.5 and 10.0.
 7. The method of claim 5, wherein thephosphate concentration of the phosphate solution is at or between 0.1and 1.0.
 8. The method of claim 1, wherein the apatite is selected fromthe group consisting of hydroxyapatite and fluorapatite.
 9. The methodof claim 1, wherein the apatite is ceramic hydroxyapatite or ceramicfluorapatite.
 10. The method of claim 1, wherein the apatite is anon-ceramic apatite.
 11. The method of claim 1, wherein the targetmolecule is a protein.
 12. The method of claim 11, wherein the proteinis selected from an antibody, an antibody fragment, and a recombinantprotein.
 13. The method of claim 1, wherein the contacting comprisescontacting the solid surface with a solution at a pH of between 5.0 and7.5.
 14. The method of claim 1, wherein the apatite solid support is inthe form of a column.